11alpha, 17alpha, 21-trihydroxy-4-pregnene-3, 20-dione and esters thereof



United States Patent 11a,17a,2l-TRIHYDROXY-4-PREGNENE-3,20-DIONE ANDESTERS THEREOF Herbert C. Murray, Hickory Corners, and Durey H.Peterson, Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo,Mich., a corporation of Michigan No Drawing. Application April 10, 1952Serial No. 281,656

18 Claims. c1. 260-39745) This invention relates to a new compound,11u,17a,21- trihydroxy-4-pregnene-3,20-dione (A -pregnane-11m,17a-21-triol-3,20-dione), and to novel esters thereof and a process for theproduction of the same.

The novel compound of the invention has the following and is useful inproducing esters according to the present invention which esters arereadily purifiable forms and have utility in the synthesis of esters ofcortisone.

It is an object of the invention to provide a novel compound11a,17a,21-trihydroxy-4-pregnene-3 ,20-dione.

. Another object is the provision of novel esters thereof.

A further object is provision of a process for making esters of'11oz,170:,21-trihydroXy-4-pregnene-3,20-dione. Other objects will beapparent to those skilled in the art to which the invention pertains.

The novel esters of the invention are prepared from the novel1la,17a,21-trihydroxy-4-pregnene-3,ZO-dione which in turn may beprepared from 17a,21-dihydroxy- 4-pregnene-3,20-di0ne and the estersthereof by the oxygenating action of a culture of fungus of the orderMucorales as described in our applications of which this application isa continuation-in-part, Serial No. 180,496, filed August 19, 1950 (nowabandoned), and Serial No. 272,944, filed February 23, 1952 (now Patent2,602,769), or as described in the preparations following in thisspecification.

The microbiological oxygenation is carried out with the employment of anoxygenating fungus which is a species of a genus of a family of theorder Mucorales or the oxygenating enzymes obtainable therefrom. Amongthe several families of this order, the genera of the familiesMucoraceae and Choanephoraceae are of greatest utility, and, among thegenera of these families of fungi, the Rhizopus, Cunninghamella, andMucor have been found of greatest value in the method of the presentinvention. Species of these genera which are operative in the method ofthe invention include, for example, the Rhizopus species microsporus,circinans, oligosporus, arrhizus, cohm'z', oryzae, nigricans, chinensis,japonicus, tritici, kaz nsis, delemar, shanghaiensis, reflexus, andsynonymous species, which are in fact identical with those namedalthough differently named, and the representative Mucor species mucedo,griseo cyanus, hiemalis, hiemalz's var. albus, rouxii, adventitius,christianiensis, circinelloides, dubius, genevensis, javanicus,microsporus, parasticus, gravensis, plumbeus, plumbeus var. spinesens,microsporus, ramannimms, racemosus, and the like.

2 While species of genera of families of the order Mucorales, andespecially those of the family Mucoraceae, are broadly operative in' themethod of the present invention as the oxidizing fungi, for reasons ofeconomy: and productivity, species of the genera Rhizopus and.Cunninghamella are preferably utilized for production of optimum yieldsof 11,17a,21-trihydroxy-4-pregnene-.

3,20-dione, although in certain cases under particular circumstances theother genera and other species may,

be most advantageously employed. Species of these mentioned genera,however, in particular demonstrate pronounced steroid oxygenatingactivity, and are thereby able, according to the method of theinvention, when suitably grown and contacted with 17a,21-dihydroxy-4-'pregnene-3,20-dione, to accomplish oxygenation of the eleven position ofthe steroid on anindustrially important as identified in H. Zycha,Kryptogamenfiora der Mark A. glauca), 'Spinellus (S. sphaerosporus),Phycomyces I Brandenburg, Band Via, l-264 (1934) include Parasitella (Psimplex), Zygorhynchus (Z. heterogamus, Z. moelleri), Circinella (C.spinosa), Actinomucor (A. repens'), Pirella (P. circinans), Absidia (A.reflexa,

(Ph. blakesleeanus, Ph. theobromatus), Sporodinia (Sp. grandis), Pilaira(P. anomala), Pillobolus (P. crystallinus), Dicoccum (D. asperum),Tieghemella (T. orchidities, T. italica).v Other Mucorales families withtheir genera and representative species include Thamnidiaceae:Thamnidium (Th. elegans), Dicranophora (D. fulva), Chaetostylum (C.fresenii), Helicostylum (H. piriforme), Chaetocladium (Ch. brefeldii);Choanephoraceae: Blakeslea (B. trispora), Choanephora (Ch.cucurbitarum), Rhopalomyces (Rh. elegans), Cunninghamella (C. elegans,C. verticulata), Thamnocophalis (Th. quadrupedata), Mycotypha (M.microspom); Cepalidaceae: Piptocephalis (P. preseniana), Syncephalis (S.reflexa, S. nodosa),

Spinalia (Sp. radians), Syncephalastrum (Saracemosum, S. fulvium),Dispira (D. comma), Coomensia (C. pectinata), Kickxella (K.alabastrina); Mortierellaceae: Mortierella (M. pusilla, M. alpina),Haplosporangium (H. bisporale), Dissophora (D. decumbens); Endogenaceae:Endogone (E. reniformis), (S. coromiodes), Glaziella (G. vesiculasa).

Of the Rhizopus genus, species of which, as stated previously, arepreferred in the methodof the invention, according to H. Zycha, Krypt.der Mark Brandenburg Band Vla, 120 (1935), many commonly known speciesare synonymous. Thus Rhizopus microsporus may be known as Rh. minimus,Mucor speciosus, Rh. speciosus, and Rh.. equinus; Rhizopus circinas maybe known as Rh. reflexus; Rhizopus oligosporus may be known as Rh.delmar or Rh. tamari; Rhizopus arrhizus may be known as Rh. nodosus, Rh.ramozus, Rh. maydis M ucor arrhizus, Mucor norvegicus, Rh. pusillus, Rh.bovinus, Rh. Cambodia, Rh. chinensis and Rh. t'riticz'; Rhizopus cohniimay be known as Rh. suilius; Rhizopus oryzae may be known as Rh.japonicus or Rh. tonkinensis; Rhizopus nigricans may be known as Mucorst0l0nif er, Rh. niger, Rh. artocarpi, Mucor niger, Rh. nigricansSclerocystis 3 vars. minor, or Rh. nigricans var. luxurious; andRhizopus echinatus is itself a doubtful species which may be synonymouswith Rhizopus nigricans.

Of the Cunninghamella genus, the species, C. echinulam; C. bainieri, C.blakesl'eeana (ATCC 9245), C. elegans, C. verticulata, and C.bert/wllitiae, among others, have been used. These Cunninghamellaspecies and the Mucor javanicus, Mucor adventitious atu'anticus, Mucormicrosporus (ATCC 8541) also demonstrate, in accordance with ourprocesses, the oxygenation of l7a,2l-dihydroxy-4-pregnene-3,20-dione.

The selected species of fungus of the order Mucorales is suitably grownon a medium containing available car bon, illustratively carbohydratessuch as sugars or starches; assimilable nitrogen, illustratively solubleor insoluble proteins, peptones or amino acids; and mineralconstituents,- illust-ratively phosphates and magnesium sulfate; andother art recognized, desirable-or adventitious, additions. Themediummay desirably have a pH before inoculationof between about 4.5 and 5.9although a higher or lower pH may be used.

Inoculation of the fungal growth-supporting medium with'the selectedfungus of the Mucorales order may be accomplished in any suitablemanner. Growth of the fungus is readily promoted by maintainingincubation temperatures of about room temperature, e. g. twenty totwenty-eight degrees centigrade, but a relatively wide range oftemperature is suitable.

The period of fungal growth required before the170:,21-dihydroxy-4-pregnene-3,ZO-dione is exposed to the oxygenatingactivity of the fungus does not appear to be critical. For example, the17u,21-dihydroxy-4- pregnene-3,20-dione can be added either beforesterilization of the medium, at the time of inoculating the medium withthe selected Mucorales species, or at some time, e. g. 24 to 48 hours,later. The 17a,21-dihydroxy-4- pregnene-3,20-dione to be oxygenated maybe added at any desired concentration although for practical reasonssteroid substrate at a concentration of about 0.01 gram to 0.25 gram orup to about 0.6 gramper liter of media or even 0.8 gram per liter issatisfactory although higher concentrations may be used with someinhibition of oxygenating activity. The addition of steroid substrate tobe oxygenated may be accomplished in any suitable manner so as topromote intimate contact of the steroid substrate with the oxygenatingactivity of the fungus and/ or fungal enzymes, such as by dispersing thesteroid substrate alone, with a dispersing agent, or in water-miscibleorganic solvent solution by mixing or homogenizing with the fungalmedium to form a suspension or dispersion of'fine steroid particles.Either submerged or surface culture procedures may be employed withfacility, although submerged culture is preferred. Alternatively. theoxygenating activity or oxygenating enzymes of a growth of the fungusmay be separated from the fungus or the medium, admixed with the steroidor a solution or suspension thereof, and the mixture subjected toaerobic conditions to accomplish oxygenation of the steroid.

The temperature employed during the period of oxygenation of the steroidneed be maintained only within such ranges as support life, activegrowth or the oxygenating activity of the fungal organism.

While any form of aerobic incubation is satisfactory for the growth ofthe selected fungus or the bio-oxygenation of the steroid substrate, theefiiciency of oxygenation is related to aeration. Therefore, controlledaeration, as by agitation and/or blowing air through the fermentationmedium is usually employed.

The time required for the bio-oxygenation of the steroid variesaccording to the results desired. When the steroid substrate is presentat the time of inoculation of the medium, periods of from eight to72hours may be used. However, when the steroid is added to the fungus,fungal medium, fungal enzymes, or the fermentation liquor aftersubstantial aerobic growth of the fungal organism, e. g.,. after. 16 to24 hours, the. conversionv of the steroid substrate begins immediatelyand high yields are obtained in from one to 72 hours, 24 hours beingsatisfactory. In lieu of direct contact of the steroid to be oxygenatedwith the fermenting media containing viable Mucorales fungi, the steroidmay be oxygenated by contact with the fermented liquor, free of fungi,or with the fungi or enzymes of Mucorales preferably in the presence ofan aqueous menstruum, or with the enzymes obtained from the fungi.

After completion of the oxygenating fermentation reaction, the resulting11,17a,2l-trihydroxy-4-pregnene- 3,20-dioneis recovered from thefermentation reaction mixture. An especially advantageous manner ofrecovering the 11,17a, 2l-trihydroxy-4-pregnenea3,ZO-dione involvesextracting the fermentation reaction mixture, including the fermentationliquor and mycelia in cases where the steroid is added'directlyto theculture, with a water-immiscible organic solvent, e. g. methylenechloride, ether, amyl acetate, and the like. The fermentation liquor andmycelia may be separated and separately extracted with suitablesolvents. The combined extracts are desirably washed with mild alkalinesolutions, and water, dried over anhydrous sodium sulfate, freed ofsolvent and purified by recrystallization from organic solvents or bychromatography.

The thus-produced 11,l7u,2l-trihydroxy-4-pregnene- 3,20-dione crudefermentation product free of aqueous phase contaminants may be eitherpurified and separated or directly, and without prior purification orseparation, reacted with an acylating agent such as, for example,ketene, a ketene of a selected acid, an acid, acid chloride, acidanhydride, or other known acylating agent, usually in a solvent,illustratively benzene, toluene, or ether, and maintained at atemperature between about zero degrees centigrade and the boiling pointof the reaction mixture, suitably at about room temperature, for aperiod of time between about one-half hour and about hours. The time ofreaction as Well as the temperature atwhich the reaction is performed,the acylating agent, and the ratio of reactants may be varied. Thereaction mixture may then be cooled or diluted by mixing with ice orcold Water, and the product collected in an appropriate solvent which isthereafter Washed with successive portions of a mildly basic solutionand water to obtain a solution of the product which is essentiallyneutral. After drying the solvent, theacylated 11,17a,21-trihydroxy-4-pregnene- 3, 2 0dione may be isolated by evaporationof the solvent,

and the residual product may be either directly oxidized to2l-acyloxy-l7m-hydroxy-4-pregnene-3,11,20-trione or it may be firstpurified by conventional procedures, such as, for example, byrecrystallization or chromatographic purification.

The thus-described acylation process, as illustrated in greater detailin the examples following in this specification, produces both the monoesters and the di-esters, although in different proportions dependingupon the proportions of acylating agent to ll,l7ot,2l-trihydroxy-4-pregnene-3,20-dione. Using approximately one equivalent of acylatingagent to said steroid produces predominantly the mono-acylated product,whereas with about two or more equivalents of acylating agent to saidsteroid, the diacylatcd product has a greater tendency to form.

Either the crude or purifiedZI-acyloxy-llJh-dihydroxy-4-pregnene-3,20-dione maybe oxidized with anoxidizing agent, illustratively chromium trioxide, to a 21- acyloxyester of cortisone 21-acyloxy-17a-hydroxy-4- pregnene- 3,1l,20-trione)the production of which is an additional object of the present inventionand is shown in the examples.

The following preparations and examples are illustrative of the processand products of the present invention and arenot to be construed aslimiting.

PREPARATION 1 g V 110:,17e,21-trihydroxy-4-pregnene-3,20-dione u 7 Amedium was prepared of twenty grams of Edaminemillimoles per liter perhour of Na SO according tothe method of Cooper, Fernstrom and. Miller,Ind.v Eng. Chem., 36, 504 (1944). To this medium containing a 24 hourgrowth of Rhizopus nz'gricans minus strain was added two grams of17ot,21-dihy.droxy-4-pregnene-3,20- dione (Reichsteins Substance S) in50lmilliliters of acetone to provide a suspension of the steroid inlth'eculture. After an additional 96 hour period of incubation under the sameconditions of temperature and aeration, the beer and mycelium wereextracted. The myceliumwas filtered, washed twice, each time with avolume of acetone approximately equal to the volume of the mycelium andextracted twice, each time with a volume of methylene chlorideapproximately. equal to the volume of the mycelium. The acetone andmethylene chloride extracts including solvent were added to the beerfiltrate. The mixed extracts and beer filtrate were extractedsuccessively with two one-half by volume portions of methylene chlorideand then with two one-fourth by volume portions of methylene chloride.The combined methylene chloride extracts were washed with two one-tenthby volume portions of a two percent aqueous solution: of sodiumbicarbonate and then with two one-tenth by volume portions of water.After drying the methylenechloride with about three to five grams ofanhydrous sodium sulfate per liter of solvent and filtering, the solventwas removed by distillation. The semi-crystalline residue weighing 4.988grams was triturated four times, each time with six milliilters of icecold methylene chloride. Remaining insoluble was 630 milligrams ofcrystals melting .at 194 to 199 degrees centigrade. Recrystallization ofthis with a mixture of three milliliters of methanol and ten millilitersof ether resulted in-404 milligrams of crystals ',PREPARATIONZ...a...11u,17u,21-trihydroxy-4-pregnene-3,20-di0ne In the same manner as inPreparation 1, flat-hydroxy- 21-acetoxy-4-pregnene-3,20-dione isconverted to 1la,l 7a, 21-trihydroxy-4-pregnene-3,20-dioner by theaction of Rhizopus nigricans.

PREPARATION 3 1 1,8,1 75,21-trihytlfoxy-4=pregnene-3,20-dion A mediumwas prepared from-0.5 percent peptone, two percent dextrose, 0.5percent, soybean meal, 0.5 percent KH PO 0.5 percent sodium chloride and,0.3 percent.

yeast extract in tap water. To 200 milliilters of'this sterilized mediumwas added an inoculum of the vegetative mycelia' of Cunninghamellablakesleeana; The spores had first been transferred from aspore slant toa broth' medium and-the broth medium was aerobically incubated at 24degrees centigrade for 24 to 72 hours in a recipro cating shaker-untilthe development of'vegetativegrowth.

. added 66 milligrams of compound S, ll-desoxy-l7-hy melting at 205 to210 degrees centigrade. Two recrystallizations from the same solventsabove gave 132 milli- 6 The inoculated medium containing addedvegetative my" celia of Cunninghamella blakesleeana was incubated for 48hours at 24 degrees centigrade following which was droxycorticosteronein solution in a minimum of ethanol and incubation was maintained forseven hours at 24 degrees centigrade. The beer containing steroid wasdiluted with 800 milliliters of acetone, shaken one hour on areciprocating shaker and filtered. The cake was suspended in 500milliliters of acetone, shaken another hour and again filtered. Thefiltrates were combined and the acetone was volatilized under reducedpressure at fifty degrees centigrade. Acetone was then added, ifnecessary, to bring the concentration to twenty percent acetone and thisresulting aqueous acetone solution was extracted five times each withone-third volume of Skellysolve B petroleum ether to remove fattymaterials. These extracts were back washed two times with one-tenthvolume of twenty percent aqueous acetone and the washings were added tothe main acetone extract. were extracted six times with one-fourthvolumes. of ethylene dichloride and the ethylene dichloride extract wasevaporated under vacuum to leave the steroid residue. This steroidresidue was taken up in a minimum of methylene chloride and applied tothe top of a column packed with thirty grams of silica which had beenpreviously triturated with 21 milliliters of ethylene glycol. Thenvarious developing mixtures, saturated with ethylene glycol, were passedover the column. Cuts were made as each steroid was eluted as determinedby the lowering of the absorption of light at 240 m in accordance withTable I on the automatic .chromatographic fraction cutter.

TABLE I.AUTOMATIC CHROMATOGRAPHIG FRACTION CUTTER Tube N0. Crude BandSolvent (60 milli- Solids,

liters) Milligrams cyclohexane 1-4 1t cyclohexane-methylene chloride 3:1.. 5-13 6. 4

cyclohexane-methylene chloride l:1 14-16 3.0 .cyclohcxane-met'nylcnechloride 2: 3-- 1723 6. O cyclohexane-methylene chloride 1 4" 24-38 12.2 methylene chloride 39-59 4. 8

A 7.7 milligram portion of Band 5 was taken up in a minimum of acetoneand refrigerated until crystals separated. This cold acetone mixture wascentrifuged and the supernatant liquid removed by pipette. To theremaining crystals, a few drops of ice-cold ether-acetone, three to onemixture, were added, shaken, recentrifuged and the supernatant washliquid removed by pipette. The

I PREPARATION 4 115,1 7ot,21-trihydr0xy-4-pregnene-3,20-di0ne The sporesof Cunninghamella blakesleeamz were transferred from an agar slant to abroth medium and aerobically incubated at 24 degrees centigrade for 24to 72 hours in a reciprocating shaker until the development ofvegetative growth.

Six hundred milliliters of this vegetative growth was used to inoculatetwelve liters of a sterile medium con- 1 taining grams of dextrin, 480grams of corn steep" solid, twelve grams of calcium carbonate and sixtygrams of sodium chloride which was made to twelve liters with tap waterand adjusted to pH 6.5 with sodium hydroxide before sterilization. Theinoculated medium was 'incubated at 26 degrees centigrade ina fivegallon bottle with stirring and aeration'for'48 hours.

The combined acetone extracts The seed thus prepared. wasaddediaseptically. to240 liters offa sterile medium prepared 'from- 0.5.percent. soy.- bean meaLtwo percentdextrose, 0.5 percent dried.brewers'yeast, 0. percent. sodium chloride, 0.5 percent KH PO made to volumewith tap, water and adjusted to pH 6.4. Fifty, cubic. feet of air perhour was passed through the stirredmedium for. seventeen hours at whichtime the pH was 6.1. Then twelve grams of l7ot,2l-dihy.-droxy-4-pregnene-3,ZO-dione in eight liters of redistilled ethanol whichhad been sterilized by ultrafine. sintered glass filtration was addedand the incubation continued for 11.5 hours.

Four kilograms of diatomaceous earth was added to the beenthe mixturewas passedthrough a filter press and the cakethenv washed with fortyliters of tap water. Theclear filtrate was'extracted withthree fiftyliter portionsof ethylene dichloride. The extract was then concentratedunder vacuum, in a nitrogen atmosphere at fifty degrees centigrade to avolume of 600 milliliters.

Three hundred grams of magnesium silicate was poured into acetonecontained in a chromatographic column 4.5 centimeters in: diameter and36 centimeters long. The acetone was replaced with ethylene dichlorideand the column washed with two liters of ethylene dichloride. Theethylene dichloride concentrate. was added to the top of the column anddeveloped with ten liters of dry ethylene dichloride. The adsorbedsteroids were then eluted from the column with two liters of dryacetone. After removal of the acetone at fifty degrees centigrade undernitrogen, the residue was taken up in 200 milliliters of ethyl acetateand washed twice with fifty milliliters of an equal volume. mixture of atwo percent aqueous so! dium carbonate solution and a three percentaqueous sodium bicarbonate solution. The extract was then washed withfifty milliliter portions of water until the washes were neutral. Thecombined water washes, were back extracted with thirty milliliters ofethylene dichloride and this together with a further 100' milliliters ofthe EXAMPLE 1 21 -aceto xy-1 1 0a,] 7adihydroxy-4-pregnene-3,20-dione ASOD-milligram sample of l-loz,170:,21-11'ihYd1'0XY-4-pregnene3,20-'dione was dissolved in four milliliters of pyridine. Tothis" solution 155 milligrams of acetic anhydride was added dropwise.The reaction mixture was kept at room temperature 24 hours and thendiluted with seventy milliliters of ice cold water, followed byextraction twice with fifty milliliters and twice with milliliters ofmethylene dichloride. The extract was washed with two 25 milliliterportions of five percenthydrochloric' acid, with 25 milliliters of tenpercent sodium bicarbonate solution, and with water to neutrality. Theextract was dried with anhydrous sodium sulfate and the solventevaporated. The oily residue was-dissolved in 125 milliliters ofethylene dichloride and chromatographed over eight grams of Florisilsynthetic magnesium silicate. Solvents used to develop the column werein IOO-milliliter portions in the following order: 1 and 2 ethylenedichloride, 3 to 5 ethylene dichloride withacetone 25:1

ratio respectively by volume,- 6 and 7 ethylene dichloride;

with acetone 15:1 ratio, 8 to 11 ethylene dichloridewith acetone 12:1ratio, 12 to 14 ethylene dichloride with.

1 l5,17'a,21-trihydroxy-4-pregnene-3,20-dione r EXAMPLE 2 1111,21-diacet0xy -1 7a-hydroxy-4pregnene-3,20-dione Fractional extracts,number eight to eleven. inclusive,

fromthe chromatographic development in Example 1' were-freedof;solvent,aby evaporation, combined and recrystallized from-acetonebydropwise addition of petroleum ether. Three. recrystallizations yieldedmilligrams of. 1111,21-diacetoxy-l7a-hydroxy-4spregnene-3,20-

dione,.melting:atx198 to 202 degrees Centigrade, [111 ,3 ofplus.=115idegrees- (1.145 in chloroform), k equal to- 33.33.. Infraredspectrum verified the indicated structure.

Analysis.-Calculated for Cg H O C, 67.24; H; 7.67. Found: C, 67.43; H,7.94.

EXAMPLE 3 2.1 -acet0xy-1l 3,1 7a-dihydr0xy-4-pregnerte-3,20-di0ne To asolution of- 500 milligramsof11fi,17u,21-trihydroxy-4-pregnene-3,20-dione in five milliliters ofpyridine,

l'milligrams of' acetic anhydride was added dropwise; After beingmaintained at-room temperature for 24 hours, the reaction mixture wasdiluted with millilitersof icecold 'water, andthediluted mixture wasextracted twice with fifty milliliter portions of cold ether andtwice'with 25-milliliter portions of methylene chloride. The combinedextracts were washed with two 25-milliliterportions-of cold'five percenthydrochloric acid, with 25 milliliters of two percent sodiumvbicarbonate solution, and

with water to'neutrality. The washed extract was dried overanhydroussodium sulfate, filtered, and the solventevaporated.Recrystallization from acetone by the dropwise addition of petroleumether produced 2l-acetoxy- 1 113,17a-dihydroxy-4-pregnene-3,ZO-dione.

EXAMPLE. 4 21-pr0pionyl0xy-1'1m1 7a-dihydr0xy-4 pregnene-3,20-dione and1 1u,21 -dipr0pi0nyloxy-4-pregnene-3,20-di0ne Following the procedure ofExample 1, using the equivalent proportion of propionicanhydride inplace of acetic,

anhydride andseparating by; chromatography produced 21 propionyloxy:,170; dihydroxy 4 pregnene- 3,20 dione and 11a,21 dipropionyloxy 17ahydroxy- 4-pregnene-3' 2O-dione.

EXAMPLE 5 21-prapionyloxy-115,17a-dihydroxy-4-pregnene- 3,20-dioneFollowing the procedure of Example 3, using the equivalent proportionofpropionic anhydride in place of aceticanhydride, produced21-propionyloxy-llath-dihydroxy-4-pregnene-3,ZO-dione.

EXAMPLE. 6.1

21 trimethylacetoxy 11,u, 17m -.dikydroxy-4-pregnene- 3,20-a'iohe and11bt,21 di(trimethylacetoxy)-17u-hydroxypregnerte-3,20-di0ne To onegramof. 11a, 17a,21-trihydroxy-4-pregnene- 3,20-dione dissolved intenmilliliters of freshly distilled pyridine was added dropwise 300milligrams of trimethylacetyl chloride. The reaction mixture wasmaintained. at

room temperature for 24 hours and then quenched, with.

150. milliliters ofice-cold water. Extraction andchromatographicseparation, as in Example 1,. produced 21-trirnethylacetoxy-l1,17a-dihydroxy 4 pregnene 3,20-

dione and 116:,21-di(trimethylacetoxy)-17a-hydroxy-4-pregnene-3,20-dione.

EXAMPLE 7 l 21 ,6 cyclopenzylpropionyloxy 11,17a dihydroxy- 4 pregnene3,20 dione and 11,21 di 7 pi cyclopemylpropionyloxy 17o: hydroxy 4pregnene- 3,20-dine EXAMPLE 8 21 benzoxy 1101,1711 dihydroxy 4 pregnene3,20-

dione and 110:,21 dibenzoxy 17oz hydroxy 4- pregnene 3,20 dioneFollowing the procedure of Example 6, using the equivalent proportion ofbenzoyl chloride in place of trimethylacetyl chloride produced21-benzoxy-11ot,17ot-dihydroxy 4 pregnene 3,20 dione and l1a,2ldibenzoxy-17a-hydroxy-4-pregnene-3,20-dione.

Other mono-esters and di-esters of11a,17oz,2l-trihydroxy-4-pregnene-3,20-dione and mono-esters of l18,17u-. 21-trihydroxy-4-pregnene-3,ZO-dione are prepared according tovarious acylation procedures such as illustrated in the examples, or byreaction with ketene, ketenes of selected acids, selected acids, acidanhydrides, or acid chlorides, in an organic solvent such as pyridine orthe like. Representative 21-acyloxy-l 1oz, 17a-dihydroxy-4-pregnene-3,20 diones, 110:,21 diacyloxy 17cc hydroxy 4- pregnene 3,20 diones, and21 acyloxy 115,170:- dihydroxy-4-pregnene-3,20-di0nes thus-preparedinclude one to eight carbon atom carboxylic acid acyloxy esters ofsaturated or unsaturated aliphatic, carbocyclic, cycloaliphatic, aryl,arylalkyl, alkaryl, mono, di or polycarboxylic acids which form estergroups such as, for ex-- ample, formyloxy, acetoxy, propionyloxy,dimethylacetoxy, trimethylacetoxy, butyryloxy, valeryloxy, hexanoyloxy,heptanoyloxy, octanoyloxy, benzoxy, phenylacetoxy, toluoyloxy,napthoyloxy, cyclopentylformyloxy, fl-cyclopentylpropionyloxy,acrylyloxy, cyclohexylformyloxy, the half and di-esters of malonic,maleic, succinic, glutaric and adipic acids, and the like. The acids mayalso contain non-interfering substituents, such as mono or poly halo,chloro, bromo, hydroxy, methoxy, and the like, if desired.

If a mixed ester involving two diiferent acyl groups is desired, thel1a,17,21-trihydroxy-4-pregnene-3,20-dione may be partially esterifiedwith one acylating agent and the resulting mono-ester may then becompletely esterified with an acylating agent which introduces adifferent acyl group. Thus 11e-propionyloxy-2l-B-cyclopentylpropionyloxy17a hydroxy 4 pregnene 3,20- dione or other mixed esters of the hereinmentioned acid groups may be prepared.

EXAMPLE 9 Cortisone acetate 162 milligrams of21-acetoXy-l1a,17a-dihydroxy-4- pregnene-3,20-dione, obtained in Example1, was dissolved in ten milliliters of glacial acetic acid and oxidizedby adding dropwise a solution of thirty milligrams of chromium trioxide(CrO in 0.5 milliliter of water and two milliliters of acetic acid.After six hours at room temperature, the green solution was diluted withtwenty milliliters of methanol and evaporated under reduced pressure.The crystalline residue was suspended in 25 milliliters of ten percentsodium bicarbonate solution and extracted with four 25 milliliterportions of a mixture of {our parts of ether to one part of chloroformby volume.

The extract was washed thrice with water and dried over anhydrous sodiumsulfate. 1 1

The resultant crystalline oxidation product weighed 166 milligrams.Recrystallization from acetone yielded 138.5 milligrams of compound Eacetate, 1 7a-hydroxy- 2l-acetoxy-4-pregnene-3,11,20-trione, otherwiseknown as cortisone acetate, with a melting point of 243 to 245 degreescentigrade, K 8 equal to 36.33, [(11 of plus 169 degrees (0.3657 inchloroform).

Analysis.Calculated for C33H3bO3 C, 68,61; H, 7.52. Found: C, 68.52; H,7.61.

In a similar manner, oxidizing the herein described 21 acyloxy 11,17adihydroxy 4 pregnene 3,20- diones or21-acyloxy-1118,17a-dihydroxy-4-pregnene-3,20- diones with, for example,chromium trioxide in acetic acid produces the corresponding acid estersof cortisone, 21 acyloxy 17a hydroxy-4-pregnene-3,11,20-triones whereinthe acyloxy group is the same as that in the starting 21 acyloxy 11,170:dihydroxy 4 pregnene- 3,20-diones.

It is to be understood that the invention is not to be limited to theexact details of operation or exact organisms and compounds shown anddescribed, as obvious modifications and equivalents will be apparent toone skilled in the art, and the invention is therefore to be limitedonly by the scope of the appended claims.

We claim:

1. A -pregnene-11a,17a,21-triol-3,20-dione of the formula:

2. A compound from the group consisting of 11a,-17a,21-trihydroxy-4-pregnene-3,20-dione and the esters thereof ofcarboxylic acid containing not more then ten carbon atoms selected fromthe class consisting of hydrocarbon carboxylic acids, halohydrocarboncarboxylic acids, hydroxyhydrocarbon carboxylic acids, andmethoxyhydrocarbon carboxylic acids.

3. An 1lot,l7a,21 trihydroxy-4-pregnene-3,20 dione ester of ahydrocarbon carboxylic acid containing not more than ten carbon atoms inwhich the acyl group may contain non-interfering substituents selectedfrom the class consisting of halo, hydroxy, and methoxy.

4. An 11a,17oc,21 trihydroxy-4-pregnene-3,20 dione ester of ahydrocarbon carboxylic acid containing from one to ten carbon atoms.

5. An 1loz,17cc,2l trihydroxy-4-pregnene-3,20 dione ester of ahydrocarbon carboxylic acid containing from one to eight carbon atomsinclusively and selected from the group consisting of aliphatic acidsand carbocyclic acids.

6. An ester of 11a,17a,21-trihydroxy-4-pregnene-3,20--

1 1oz,17a,21-trihydroxy-4-pregnene-3,ZO-dione.

1.$4-PREGNENE-11A,17A821-TRIOL-3,20-DIONE OF THE FORMULA: